Entrapment in phospholipid vesicles quenches photoactivity of quantum dots

Quantum dots have emerged with great promise for biological applications as fluorescent markers for immunostaining, labels for intracellular trafficking, and photosensitizers for photodynamic therapy. However, upon entry into a cell, quantum dots are trapped and their fluorescence is quenched in endocytic vesicles such as endosomes and lysosomes. In this study, the photophysical properties of quantum dots were investigated in liposomes as an in vitro vesicle model. Entrapment of quantum dots in liposomes decreases their fluorescence lifetime and intensity. Generation of free radicals by liposomal quantum dots is inhibited compared to that of free quantum dots. Nevertheless, quantum dot fluorescence lifetime and intensity increases due to photolysis of liposomes during irradiation. In addition, protein adsorption on the quantum dot surface and the acidic environment of vesicles also lead to quenching of quantum dot fluorescence, which reappears during irradiation. In conclusion, the in vitro model of phospholipid vesicles has demonstrated that those quantum dots that are fated to be entrapped in endocytic vesicles lose their fluorescence and ability to act as photosensitizers

Cellular Interaction with Polymeric Nanoparticles: The Effect of PEGylation and Monomer Composition

It is of importance to understand which nanoparticle properties that govern the interactions between particles and cells, in order to develop a nanocarrier with the desired functionality. In this thesis, nanoparticles made of biodegradable poly(alkyl cyanoacrylate), with either butyl or octyl as the side chain, and with different polyethylene glycol (PEG) surface coatings, have been utilized. The objective was to determine whether variations in these properties influenced cellular uptake and toxicity in prostatic adenocarcinoma cells, as well as the release of a model drug, nile red.Cellular uptake of nanoparticles was investigated in vitro using flow cytometry and confocal laser scanning microscopy. It was established that the encapsulated nile red marker could dissociate from the particles, thus making evaluation of cellular uptake difficult. No alterations in PEG type or chain lengths made nile red remain in the particles to such a degree that endocytosis of nanoparticles could be detected. Spectrophotometric analyses of nile red release from the nanoparticles and into cell medium demonstrated that around 45% or more of originally encapsulated nile red was released after 3 hours. This confirmed high nile red release from the particles, and at the same time it showed that changes in PEGylation did not reduce the release to any extent. After estimating PEG chain surface densities, it was evident that all particles had very low PEG densities, providing an explanation to why nile red dissociated from the particles to such a high degree: the PEG layer did not shield a large enough part of the particle surface area to effectively hinder release of nile red.Cytotoxicity after nanoparticle exposure was determined using an assay measuring the metabolic activity of cells. Toxicity was found to be strongly dependent on the length of the alkyl side chain in the monomer, where the longest chain, with the lowest degradation rate, was the least toxic. Altogether, this suggests toxicity induced by release of degradation products, but it can also be attributed to residual surfactant from the synthesis, as the observed cytotoxicity was higher than what is reported in literature for similar nanoparticles

Evaluation of Carrier Compounds for Systemic and Intracavitary α-Radionuclide Therapy of Cancer

Radionuclide therapy utilizing an α-emitting radioisotope combined with a carrier compound is a promising therapeutic strategy to combat micrometastases in cancer patients. The purpose of the carrier is to deliver the radiation to the tumor site so that the cancer cells are irradiated, become damaged and eventually die. Treatment of microsized tumors with α-emitting radionuclides has several advantages. The short range of α-particles in tissue allows high energy to be deposited within a distance corresponding to the size of the micrometastases, resulting in efficient cell kill and limited exposure to adjacent tissues. In this thesis, two types of radiolabeled carriers have been evaluated: An anti-CD146 monoclonal antibody, named OI-3, for cell-specific targeting following systemic administration and inorganic calcium carbonate microparticles for local treatment of cavitary cancers. Both carriers were able to maintain and transport their radioactive payload to the target site. The OI-3 antibody bound selectively to human osteosarcoma cell lines in vitro and was able to target CD146 expressing tumors after intravenous injection in mice. Calcium carbonate microparticles labeled with radium-224 retained its radioactive payload to a large extent in vivo, as the radioactivity mainly remained in the peritoneal cavity of mice after intraperitoneal injection. For therapeutic purposes, the potential of combining the carriers with radium-224 or its daughter lead-212, which both generate emission of α-particles, was explored. Radium-224 labeled calcium carbonate microparticles exhibited significant antitumor effect in two murine models of human ovarian cancer. In addition, a novel method for producing antibodies labeled with lead-212 was proposed and its feasibility established. Here, a solution of radium-224 was used directly for labeling, eliminating the need to prepare a pure solution of lead-212, which is a relatively cumbersome and time-consuming process

Therapeutic effect of α-emitting 224Ra-labeled calcium carbonate microparticles in mice with intraperitoneal ovarian cancer

BACKGROUND: Ovarian cancer patients with chemotherapy-resistant residual microscopic disease in the peritoneal cavity have a considerable need for new treatment options. Alpha-emitting radionuclides injected intraperitoneally may be an attractive therapeutic option in this situation as they are highly cytotoxic, while their short range in tissues can spare surrounding radiosensitive organs in the abdomen. Herein we evaluate the therapeutic efficacy of a novel α-emitting compound specifically designed for intracavitary radiation therapy. METHODS: The α-emitter 224Ra was absorbed on calcium carbonate microparticles. Immunodeficient, athymic nude mice with human ovarian cancer cells growing intraperitoneally were treated with different activity levels of 224Ra-microparticles. Tumor growth, survival, and tolerance of the treatment were assessed. Two tumor models based on the cell lines, ES-2 and SKOV3-luc, with different growth patterns were studied. RESULTS: In both models, intraperitoneal treatment with 224Ra-microparticles gave significant antitumor effect with either considerably reduced tumor volume or a survival benefit. An advantageous discovery was that only a few kilobecquerels per mouse were needed to yield therapeutic effects. The treatment was well tolerated up to a dose of 1000 kBq/kg with no signs of acute or subacute toxicity observed. CONCLUSIONS: Intraperitoneal α-therapy with 224Ra-microparticles demonstrated a significant potential for treatment of peritoneal micrometastases in ovarian carcinoma

Preparation of 212Pb-labeled monoclonal antibody using a novel 224Ra-based generator solution

Introduction: Alpha-emitting radionuclides have gained considerable attention as payloads for cancer targeting molecules due to their high cytotoxicity. One attractive radionuclide for this purpose is 212Pb, which by itself is a β-emitter, but acts as an in vivo generator for its short-lived α-emitting daughters. The standard method of preparing 212Pb-labeled antibodies requires handling and evaporation of strong acids containing high radioactivity levels by the end user. An operationally easier and more rapid process could be useful since the 10.6 h half-life of 212Pb puts time constraints on the preparation protocol. In this study, an in situ procedure for antibody labeling with 212Pb, using a solution of the generator nuclide 224Ra, is proposed as an alternative protocol for preparing 212Pb-radioimmunoconjugates. Methods: Radium-224, the generator radionuclide of 212Pb, was extracted from its parent nuclide, 228Th. Lead-212-labeling of the TCMC-chelator conjugated monoclonal antibody trastuzumab was carried out in a solution containing 224Ra in equilibrium with progeny. Subsequently, the efficiency of separating the 212Pb-radioimmunoconjugate from 224Ra and other unconjugated daughter nuclides in the solution using either centrifugal separation or a PD-10 desalting size exclusion column was evaluated and compared. Results: Radiolabeling with 212Pb in 224Ra-solutions was more than 90% efficient after only 30 min reaction time at TCMC-trastuzumab concentrations from 0.15 mg/mL and higher. Separation of 212Pb-labeled trastuzumab from 224Ra using a PD-10 column was clearly superior to centrifugal separation. This method allowed recovery of approximately 75% of the 212Pb-antibody-conjugate in the eluate, and the remaining amount of 224Ra was only 0.9 ± 0.8% (n = 7). Conclusions: The current work demonstrates a novel method of producing 212Pb-based radioimmunoconjugates from a 224Ra-solution, which may be simpler and less time-consuming for the end user compared with the method established for use in clinical trials of 212Pb-TCMC-trastuzumab

A Novel Single-Step-Labeled 212Pb-CaCO3 Microparticle for Internal Alpha Therapy: Preparation, Stability, and Preclinical Data from Mice

Lead-212 is recognized as a promising radionuclide for targeted alpha therapy for tumors. Many studies of 212Pb-labeling of various biomolecules through bifunctional chelators have been conducted. Another approach to exploiting the cytotoxic effect is coupling the radionuclide to a microparticle acting as a carrier vehicle, which could be used for treating disseminated cancers in body cavities. Calcium carbonate may represent a suitable material, as it is biocompatible, biodegradable, and easy to synthesize. In this work, we explored 212Pb-labeling of various CaCO3 microparticles and developed a protocol that can be straightforwardly implemented by clinicians. Vaterite microparticles stabilized by pamidronate were effective as 212Pb carriers; labeling yields of ≥98% were achieved, and 212Pb was strongly retained by the particles in an in vitro stability assessment. Moreover, the amounts of 212Pb reaching the kidneys, liver, spleen, and skeleton of mice following intraperitoneal (i.p.) administration were very low compared to i.p. injection of unbound 212Pb2+, indicating that CaCO3-bound 212Pb exhibited stability when administered intraperitoneally. Therapeutic efficacy was observed in a model of i.p. ovarian cancer for all the tested doses, ranging from 63 to 430 kBq per mouse. Lead-212-labeled CaCO3 microparticles represent a promising candidate for treating intracavitary cancers

Contact-mediated intracellular delivery of hydrophobic drugs from polymeric nanoparticles

Encapsulation of drugs in nanoparticles can enhance the accumulation of drugs in tumours, reduce toxicity toward healthy tissue, and improve pharmacokinetics compared to administration of free drug. To achieve efficient delivery and release of drugs at the target site, mechanisms of interaction between the nanoparticles and cells and the mechanism of delivery of the encapsulated drug are crucial to understand. Our aim was to determine the mechanisms for cellular uptake of a fluorescent hydrophobic model drug from poly(butylcyanoacrylate) nanoparticles. Prostate adenocarcinoma cells were incubated with Nile Red-loaded nanoparticles or free Nile Red. Uptake and intracellular distribution were evaluated by flow cytometry and confocal laser scanning microscopy. The nanoparticles mediated a higher intracellular level and more rapid uptake of encapsulated Nile Red compared to model drug administered alone. The main mechanism for delivery was not by endocytosis of nanoparticles but by nanoparticle-cell contact-mediated transfer directly to the cytosol and, to a smaller extent, release of payload from nanoparticles into the medium followed by diffusion into cells. The payload thus avoids entering the endocytic pathway, evading lysosomal degradation and instead gains direct access to intracellular targets. The nanoparticles are promising tools for efficient intracellular delivery of hydrophobic anticancer drugs; therefore, they are clinically relevant for improved cancer therapy

Radon-220 diffusion from 224Ra-labeled calcium carbonate microparticles: Some implications for radiotherapeutic use.

Alpha-particle emitting radionuclides continue to be the subject of medical research because of their high energy and short range of action that facilitate effective cancer therapies. Radium-224 (224Ra) is one such candidate that has been considered for use in combating micrometastatic disease. In our prior studies, a suspension of 224Ra-labeled calcium carbonate (CaCO3) microparticles was designed as a local therapy for disseminated cancers in the peritoneal cavity. The progenies of 224Ra, of which radon-220 (220Rn) is the first, together contribute three of the four alpha particles in the decay chain. The proximity of the progenies to the delivery site at the time of decay of the 224Ra-CaCO3 microparticles can impact its therapeutic efficacy. In this study, we show that the diffusion of 220Rn was reduced in labeled CaCO3 suspensions as compared with cationic 224Ra solutions, both in air and liquid volumes. Furthermore, free-floating lead-212 (212Pb), which is generated from released 220Rn, had the potential to be re-adsorbed onto CaCO3 microparticles. Under conditions mimicking an in vivo environment, more than 70% of the 212Pb was adsorbed onto the CaCO3 at microparticle concentrations above 1 mg/mL. Further, the diffusion of 220Rn seemed to occur whether the microparticles were labeled by the surface adsorption of 224Ra or if the 224Ra was incorporated into the bulk of the microparticles. The therapeutic benefit of differently labeled 224Ra-CaCO3 microparticles after intraperitoneal administration was similar when examined in mice bearing intraperitoneal ovarian cancer xenografts. In conclusion, both the release of 220Rn and re-adsorption of 212Pb are features that have implications for the radiotherapeutic use of 224Ra-labeled CaCO3 microparticles. The release of 220Rn through diffusion may extend the effective range of alpha-particle dose deposition, and the re-adsorption of the longer lived 212Pb onto the CaCO3 microparticles may enhance the retention of this nuclide in the peritoneal cavity